Vaccination of Alzheimer's model mice with Abeta derivative in alum adjuvant reduces Abeta burden without microhemorrhages

Abstract

Immunotherapy holds great promise for Alzheimer's disease (AD) and other conformational disorders but certain adverse reactions need to be overcome. The meningoencephalitis observed in the first AD vaccination trial was likely related to excessive cell-mediated immunity caused by the immunogen, amyloid-beta (Abeta) 1-42, and the adjuvant, QS-21. To avoid this toxicity, we have been using Abeta derivatives in alum adjuvant that promotes humoral immunity. Other potential side effects of immunotherapy are increased vascular amyloid and associated microhemorrhages that may be related to rapid clearance of parenchymal amyloid. Here, we determined if our immunization strategy was associated with this form of toxicity, and if the therapeutic effect was age-dependent. Tg2576 mice and wild-type littermates were immunized from 11 or 19 months and their behaviour evaluated prior to killing at 24 months. Subsequently, plaque- and vascular-Abeta burden, Abeta levels and associated pathology was assessed. The therapy started at the cusp of amyloidosis reduced cortical Abeta deposit burden by 31% and Abeta levels by 30-37%, which was associated with cognitive improvements. In contrast, treatment from 19 months, when pathology is well established, was not immunogenic and therefore did not reduce Abeta burden or improve cognition. Significantly, the immunotherapy in the 11-24 months treatment group, that reduced Abeta burden, did not increase cerebral bleeding or vascular Abeta deposits in contrast to several Abeta antibody studies. These findings indicate that our approach age-dependently improves cognition and reduces Abeta burden when used with an adjuvant suitable for humans, without increasing vascular Abeta deposits or microhemorrhages.

Fig. 1

Antibody levels and brain amyloid burden. Groups were treated from 11 to 24 months (A and C) and from 19 to 24 months (B and D). (A) 11–24 months. K6Aβ1–30 in alum adjuvant elicits a good antibody response that is maintained (T1, 13 months; Tfinal, 24 months). The antibodies recognize both the immunogen and Aβ1–40. Vehicle-treated controls have some autoantibodies at old age. Treatment and antibody type measured (IgG) is indicated in the title. The x-axis depicts which peptide (coated on ELISA plate) the antibodies are recognizing. The y-axis depicts the absorbance at 450 nm. (B) 19–24 months. K6Aβ1–30 in alum adjuvant is not immunogenic when treatment is started at old age (19 months). (C) 11–24 months. Cortical amyloid burden (parenchymal and vascular) was reduced by 31% (P < 0.05) in Tg2576 mice immunized with K6Aβ1–30 in alum adjuvant from 11 to 24 months of age, compared to control mice. When analysed separately, vascular amyloid burden was not significantly altered by the immunotherapy. Total brain Aβ levels (Aβ1–40, 30% reduction, P = 0.03; Aβ1–42, 37% reduction, P = 0.02) were reduced to a similar extent as total amyloid burden. Levels of soluble Aβ were not significantly affected although soluble Aβ1–42 was reduced by 32% in the immunized mice (P = 0.08). (D) 19–24 months. No significant difference in cortical amyloid burden was observed between the immunized and nonimmunized Tg mice (P = 0.206). The error bars are standard error of the mean that applies also to all subsequent figures.

Fig. 2

Histology of plaques and microhemorrhages. (A and B) Immunohistochemistry (6E10) of Aβ plaques revealed that compared to controls (A), immunized mice (B) had fewer large plaques and fewer diffuse plaques of all sizes. This observation fits with the 31% reduction in cortical amyloid burden (see Fig. 1A). A and B are representative coronal sections (original magnification, ×50) through the retrosplenial cortex (cx) and the hippocampus (hip). (C and D) Cerebral microhemorrhages were often observed in Tg2576 mouse brain sections stained with Perls’ stain for ferric iron in haemosiderin (blue), both in controls (C) and immunized mice (D). The immunotherapy did not increase the extent of the bleeding as quantified in E and F. C and D are representative coronal sections (original magnification, ×50) through the parietal cortex of Tg2576 mice. (E) 11–24 months. Immunization with K6Aβ1–30 did not increase brain microhemorrhages in Tg2576 mice but the Tg animals had significantly more iron positive profiles per brain section compared to wild-type (Wt) littermates (Tg control vs. Wt, P < 0.01; Tg K6Aβ1–30 vs. Wt, P < 0.05). (F) 19–24 months. No significant difference was seen in brain microhemorrhages between immunized and control Tg mice.

Fig. 3

Microgliosis. The semiquantitative analysis of microglial staining with tomatolectin (Table 2) indicated more extensive microgliosis associated with Aβ plaques in the immunized animals in the 11–24 months group compared to transgenic controls (P < 0.05). No apparent differences were observed between the groups in the 19–24-month study (data not shown). (A–D) Representative images of the microglia quantified in Table 2. Original magnification ×200. In sections with +++ rating, ramified and phagocytic microglia were prominent throughout the Aβ deposit (A). Microglial involvement with the Aβ deposits was less in sections rated as ++ (B) and + (C) and virtually absent in sections rated as 0 (D).

Fig. 4

Radial arm maze. (A) 11–24 months. The Tg mice immunized with K6Aβ1–30 navigated the radial arm maze with fewer errors than control Tg mice, and performed as well as their wild-type (Wt) littermates (group effect P < 0.001; Tg control vs. Tg K6Aβ1–30 P = 0.01; Tg control vs. Wt mice P = 0.001). (B) 19–24 months. The Tg mice immunized with K6Aβ1–30 did not differ significantly from their control Tg mice in their performance in the radial arm maze.

Fig. 5

Closed field symmetrical maze. (A–G) 11–24 months. Overall, the Tg controls performed significantly worse than the immunized Tg mice (P < 0.001) and the wild-type mice (P < 0.0001), whereas the immunized Tg animals performed as well as their wild-type (Wt) littermates. Significant differences in the number of errors were observed in Maze 3 (C, Tg control vs. Wt, P < 0.05) and Maze 7 (G, Tg control vs. Tg K6Aβ1–30, P < 0.001; Tg control vs. Wt, P < 0.0001; Tg K6Aβ1–30 vs. Wt, P < 0.01). Maze 7 had the most difficult navigation pattern and Maze 3 was the second most difficult maze, as shown by the total number of errors committed (see y-axis). A strong trend for significance was observed in two other relatively complex mazes (Mazes 1 and 6) and in these mazes the Tg K6Aβ1–30 mice had similar number of errors as Wt mice. The Tg mice immunized from 19 to 24 months did not differ significantly from their Tg controls in any of the mazes (data not shown).

Fig. 6

Locomotor activity. (A) 11–24 months. Both the Tg groups were more active than their wild-type (Wt) littermates but the immunized and control Tg groups did not differ significantly in any of the parameters measured. Distance travelled (P < 0.01, Wt vs. Tg control, P < 0.01; Wt vs. Tg K6Aβ1–30, P < 0.05). Maximum speed (V_max) obtained did not differ between the groups. Average speed (V_mean, P < 0.01, Wt vs. Tg control, P < 0.01; Wt vs. Tg K6Aβ1–30, P < 0.05). Rest time (P < 0.001, Wt vs. Tg control, P < 0.001; Wt vs. Tg K6Aβ1–30, P < 0.05). (B) 19–24 months. The Tg groups did not differ in any of the locomotor parameters measured.

Fig. 7

Traverse beam and rotarod. (A) 11–24 months. The animals differed in their performance on the traverse beam (P < 0.05, Tg K6Aβ1–30 vs. Wt, P = 0.03). As expected, the mice had fewer foot slips with more trials (P < 0.0001) and the improvement was group dependent (treatment × trials P = 0.0003). (B) 11–24 months. No group differences were observed but the animals’ performance on the rotarod improved over time as expected (P = 0.0003). (C) 19–24 months. No differences were observed between the Tg groups in their performance on the traverse beam but there was a strong overall trend for significantly fewer foot slips with more trials (P = 0.06). (D) 19–24 months. No group differences were observed on the rotarod but the animals’ performance improved over time as expected (P = 0.0023).